Acute pancreatitis is an inflammatory disease that is triggered by the premature activation of proteolytic zymogens in acinar cells. As a protective mechanism, zymogens are synthesized as inactive pro-enzymes and packaged in specialized secretory granules destined for exocytosis into the pancreatic duct. During pancreatitis, proteolytic zymogens are prematurely activated in acinar cells by aberrantly mixing with hydrolases present in lysosomes. The mixing of lysosomal hydrolases with zymogens occurs in large cytoplasmic vacuoles as a result of pathogenic alterations in acinar cell membrane trafficking. It is well established that the stress-induced expression of heat shock protein-70 (HSP70) in acini provides a natural protective effect against the pathogenic mixing of lysosomal and secretory granule membrane compartments. This proposal is designed to elucidate the mechanism by which HSP70 inhibits the intra-acinar cell activation of zymogens in preventing the on-set of acute pancreatitis. HSP70 is a molecular chaperone in the cytosol that requires a co-chaperone protein to stimulate ATPase activation and substrate binding. We have 1) identified an HSP70 co-chaperone protein called cysteine string protein (CSP) in acinar cells, 2) localized this membrane associated molecule throughout the secretory pathway and 3) developed the TAT-fusion protein system to manipulate CSP expression in acinar cells and thereby study its function. We hypothesize that CSP mediates the protective effects of HSP70 against pancreatitis by targeting HSPT0 chaperone activity from the cytosol to the secretory pathway. In Specific Aim 1, mutant forms of CSP that no longer anchor to secretory granule membranes will be overexpressed acinar cells to determine the importance of CSP in targeting HSP70 to these organelles during pancreatitis. In Specific Aim II, short interference RNAs will be used to inhibit CSP expression in pancreatic Iobules and the protective effects of HSP70 on secretagogue-induced acinar cell damage will be evaluated. These studies will allow us to directly evaluate the role of CSP in the secretory pathway of acini under physiological and pathophysiological conditions.